Identification of a Retroelement from the Resurrection Plant Boea hygrometrica That Confers Osmotic and Alkaline Tolerance in Arabidopsis thaliana Yan Zhao1., Tao Xu1., Chun-Ying Shen1, Guang-Hui Xu1, Shi-Xuan Chen1, Li-Zhen Song1,2, Mei-Jing Li1, Li-Li Wang1, Yan Zhu1, Wei-Tao Lv1, Zhi-Zhong Gong3, Chun-Ming Liu2, Xin Deng1* 1 Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, China, 2 Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China, 3 State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Abstract Functional genomic elements, including transposable elements, small RNAs and non-coding RNAs, are involved in regulation of gene expression in response to plant stress. To identify genomic elements that regulate dehydration and alkaline tolerance in Boea hygrometrica, a resurrection plant that inhabits drought and alkaline Karst areas, a genomic DNA library from B. hygrometrica was constructed and subsequently transformed into Arabidopsis using binary bacterial artificial chromosome (BIBAC) vectors. Transgenic lines were screened under osmotic and alkaline conditions, leading to the identification of Clone L1-4 that conferred osmotic and alkaline tolerance. Sequence analyses revealed that L1-4 contained a 49-kb retroelement fragment from B. hygrometrica, of which only a truncated sequence was present in L1-4 transgenic Arabidopsis plants. Additional subcloning revealed that activity resided in a 2-kb sequence, designated Osmotic and Alkaline Resistance 1 (OAR1). In addition, transgenic Arabidopsis lines carrying an OAR1-homologue also showed similar stress tolerance phenotypes. Physiological and molecular analyses demonstrated that OAR1-transgenic plants exhibited improved photochemical efficiency and membrane integrity and biomarker gene expression under both osmotic and alkaline stresses. Short transcripts that originated from OAR1 were increased under stress conditions in both B. hygrometrica and Arabidopsis carrying OAR1. The relative copy number of OAR1 was stable in transgenic Arabidopsis under stress but increased in B. hygrometrica. Taken together, our results indicated a potential role of OAR1 element in plant tolerance to osmotic and alkaline stresses, and verified the feasibility of the BIBAC transformation technique to identify functional genomic elements from physiological model species. Citation: Zhao Y, Xu T, Shen C-Y, Xu G-H, Chen S-X, et al. (2014) Identification of a Retroelement from the Resurrection Plant Boea hygrometrica That Confers Osmotic and Alkaline Tolerance in Arabidopsis thaliana. PLoS ONE 9(5): e98098. doi:10.1371/journal.pone.0098098 Editor: Manoj Prasad, National Institute of Plant Genome Research, India Received November 8, 2013; Accepted April 29, 2014; Published May 22, 2014 Copyright: ß 2014 Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Natural Science Foundation of China (31270312 and 30970431), the National High Technology Research and Development Program of China (863 Program, 2007AA021403), the National Basic Research Program of China (973 Program, 2012CB114302), and the Ministry of Agriculture of China (2009ZX08009-060B). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction resurrection plants, a category of plants that are able to tolerate full desiccation (leaf relative water content ,10%) and are viable Drought, alkaline and high calcium are major environmental after rehydration within 48 h [7–9]. factors in South-west China Karst landforms that limit plant Previously, it was reported that the thylakoid pigment-protein growth and crop productivity [1,2]. Improving crop tolerance to complexes and pigment contents were highly stable during environmental stresses is thus beneficial for both the agriculture desiccation and rehydration in B. hygrometrica, but were irreversibly and ecosystem dynamics in the region. Drought stress has been lost in the desiccated leaves of a non-resurrection Gesneriaceae intensively studied in plants, resulting in the identification of a species Chirita heterotrichia [7]. The stabilization of photosynthetic large number of genes that play a potential role in tolerance apparatus during water deficit had also been reported on mechanisms [3]. In contrast, tolerance to alkaline and high resurrection plants in Haberlea spp. and Ramando spp. [8,9], calcium stress has not been intensively studied in plants. indicating that these plants may have evolved distinct adaptive Many plant species in the Gesneriaceae family such as Boea mechanisms to cope with desiccation. Plant responses to water hygrometrica, Haberlea rhodopensis, Ramando myconi, Metape- deficit are complex, and these responses can be synergistically or trocosmea peltata, Chirita heterotricha, Oreocharis flavida, and antagonistically modified by the superimposition of other stresses Paraboea rufescens [4] are well adapted to the Karst region, and [13]. It is unknown whether the concurrent environment factors grow in shady limestone crevices where the soil is alkaline [5,6]. B. such as high calcium and alkali evoke common adaptive hygrometrica, H. rhodopensis and R. myconi, are also known as PLOS ONE | www.plosone.org 1 May 2014 | Volume 9 | Issue 5 | e98098 A Retroelement Confers Osmotic and Alkaline Tolerance mechanisms that dehydration triggers in these Gesneriaceae Arabidopsis thaliana serving as an internal reference standard. The resurrection plants. histogram of relative DNA content was obtained after flow Genome-level regulation such as chromatin modification and cytometric analysis of nuclei of B. hygrometrica and Arabidopsis, which assembly, transposable elements, small RNAs, and non-coding were isolated, stained, and analyzed simultaneously. B. hygrometrica RNAs are involved in plant stress responses [14,15].With the 2C DNA content = (Boea G1 peak mean)/(Arabidopsis G1 peak exception of CDT-1 from Craterostigma plantagineum [16–18], mean)6Arabidopsis 2C DNA content (Arabidopsis 1C = 125 Mb) genomic elements that regulate stress tolerance in resurrection [27]. plants have not been identified, which is largely due to the lack of genome sequence data and genetic analysis tools. Transformation of BIBAC clones into Agrobacterium and Binary bacterial artificial chromosome (BIBAC) vectors were Arabidopsis developed for transformation of large genomic DNA fragments The BIBAC clone was sequenced to obtain terminal sequences into plants. This technology can be used overcome the technical that were used to design primers for clone-specific designation. limitations in species where genetic transformation and genome Plasmid DNA of BIBAC clones was isolated with the alkaline lysis sequences are not available [19–21]. Therefore it became a useful method and transformed into A. tumefaciens strain GV3101 via tool for phenotype-based screening of genomic elements. For electroporation. The electroporated GV3101 clones were selected example screening of a BIBAC library from Thellungiella halophila on YEB medium containing 50 mg/L rifampincin, 50 mg/L led to the identification of a clone with a 120–130-kb insert that is kanamycin, and 35 mg/L gentamycin. Random colonies were associated with improved salt tolerance [22]. Screening of a incubated in liquid YEB medium with antibiotics (as above) for 2 BIBAC library from Leavenworthia alabamica also led to the days at 28uC with shaking at 170 rpm and confirmed by PCR identification of 84 20-kb genomic clones with phenotypic effects using primer pairs designed according to the appropriate clone- such as short fruit and aborted seeds [23]. In this study, a BIBAC specific terminal sequences. Agrobacterium clones transformed with library was constructed with B. hygrometrica genomic DNA and used L1-4 were incubated in liquid YEB medium with antibiotics (as for generation of transgenic populations. A BIBAC clone that above) at 28uC, with shaking at 170 rpm. When the OD600 value conferred osmotic or alkaline tolerance was identified, and the of the culture increased to about 0.8, 500 ml of the culture was resident functional element that might be responsible for the transferred to a 50 ml culture for continued growth. These improved osmotic and alkaline tolerance was assigned. cultures were subcultured 4 times, for approximately 10 hours for each passage. 1 mL of the first culture and 1 mL of the fifth culture Materials and Methods were used to analyze the integrity of the BIBAC DNA in Agrobacterium using the multiple-marker PCR-based method [20]. BIBAC library construction Floral-dip transformation of Arabidopsis was conducted using the B. hygrometrica plants were collected from a self-bred population Columbia ecotype [28]. Transgenic plants were confirmed by grown in green house conditions in our lab. Therefore no specific PCR amplification using the BIBAC terminal specific primers. permission was required for these collections. This study did not Homozygous lines were selected through two further rounds